Transcript Nucleic Acids - Farmasi Unand

10.13.5 Antisense Drugs
• The concept of antisense compounds or
sequence-defined oligonucleotides
(ONs) offers a new specific approach to
designing drugs that target nucleic
acids,
• The idea underlying this approach is
that the antisense compound contains
the sequence of complementary bases
to those found in a short section of the
target nucleic acid.
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• This section is usually part of the
genetic message being carried by an
mRNA molecule.
• The antisense compound binds to this
section by hydrogen bonding between
the complementary base pairs.
• This inhibits translation of the message
carried by the mRNA, which inhibits the
production of a specific protein
responsible for a disease state in a
patient.
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• Antisense compounds were originally short
•
lengths of nucleic acid chains that had base
sequences that were complementary to those
found in their target RNA.
These short lengths of nucleic acid antisense
compounds were found to be unsuitable as
drugs because of poor binding to the target
site and short half-lives due to enzyme action.
However, they provided lead compounds for
further development (Figure 10.35).
Development is currently taking three basic
routes:
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flg.re fl The bleomycin’. The drug bleomycin sulphate is a mixture of a
number of bleomycins.
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• (I) modification of the backbone linking the bases to
•
•
increase resistance to enzymatic hydrolysis:
(ii) changing the nature of the sugar residue by either
replacing some of the free hydroxy groups by other
substituents or forming derivatives of these groups(iii)
modifying the nature of the substituent groups of the
bases.
Antisense compounds are able to bind to both RNA and
DNA. In the latter case they form a triple helix. At
present, antisense drugs are still in the early stages of
their development but the concept has aroused
considerable interest in the pharmaceutical industr
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• Antisense compounds are able to bind to
both RNA and DNA. In the latter case
they form a triple helix.
• At present, antisense drugs are still in
the early stages of their development
but the concept has aroused
considerable interest in the
pharmaceutical industriy
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10.13.6 Chain-cleaving Agents
• The interaction of chain-cleaving
agents with DNA results in the
breaking of the nucleic acid into
fragments.
• Currently, the main cleaving agents
are the bleomycins (Figure 10.36)
and their analogues.
• However, other classes of drug are
in the development stage.
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• The bleomycins are a group of naturally
occurring glycoproteins that exhibit
antitumour activity.
• When administered to patients they
tend to accumulate in the squamous cells
and so are useful for treating cancers
of the head, neck and genitalia.
However, the bleomycins cause pain and
ulceration of areas of skin that contain
a high concentration of keratin, as well
as other unwanted side effects.
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• The action of the bleomycins is not fully
understood. It is believed that the
bithia role moiet (domain X in Figure
10.36) intercalates with the DNA.
• In bleomycin A3 the resulting adduct to
the receptor of the host cell the virus—
receptor complex is transported into
the cell by receptor-mediated
endocytosis (see section 4.3.6).
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• In the course of this process the
protein capsid and any lipoprotein
envelopes may be removed.
• Once it has entered the host cell the
viral nucleic acid is able to use the
host’s cellular machinery to synthesise
the nucleic acids and proteins required
to produce a number of new viruses
(Figure 10.38).
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14. Viruses
• Viruses are infective agents that are
considerably smaller than bacteria.
They are essentially packages, known as
virions, of chemicals that invade host
cells.
• However, viruses are not independent
and can only penetrate a host cell that
can satisfy the specific needs of that
virus.
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• The mode of penetration varies
considerably from virus to virus. Once
inside the host cell viruses take over
the metabolic machinery of the host
and use it to produce more viruses.
Replication is often lethal to the host
cell, which may undergo lysis to release
the progeny of the virus.
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• However, in some cases the virus
may integrate into the host
chromosome and become dormant.
The ability of viruses to reproduce
means that they can be regarded as
being on the borderline of being
living organisms.
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14.1. Structure and replication
• Viruses consist of a core of either
DNA or, as in the majority of cases,
RNA fully or partially covered by a
protein coating known as the capsid.
The capsid consists of a number of
polypeptide molecules known as
capsomers (Fig.10.43).
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Figure 10.37. (a) Schematic representations of the
structure of a virus (a) without a lipoprotein
envelope (naked virus) and (h) with a lipoprotein
envelope.
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• The capsid that surrounds most viruses
consists of a number of different
capsomers although some viruses will
have capsids that only contain one type
of capsomer. It is the arrangement of
the capsomers around the nucleic acid
that determines the overall shape of
the virion.
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• In the majority of viruses, the
capsomers form a layer or several
layers that completely surround the
nucleic acids. However, there are
some viruses in which the
capsomers form an open-ended
tube that holds the nucleic acids.
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• In many viruses the capsid is coated
with a protein-containing lipid bilayer
membrane.
• These are known as enveloped viruses.
Their lipid bilayers are often derived
from the plasma membrane of the host
cell and are formed when the virus
leaves the host cell by a process known
as budding.
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• Budding is a mechanism by which a virus
leaves a host cell without killing that
cell. It provides the virus with a
membrane whose lipid components are
identical to those of the host (Fig.
10.43). This allows the virus to
penetrate new host cells without
activating the host’s, immune systems.
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• Viruses bind to host cells at specific
receptor sites on the host’s cell
envelope.
• The binding sites on the virus are
polypeptides in its capsid or lipoprotein
envelope. Once the virus has bound to
the receptor of the host cell the virus–
receptor complex is transported into
the cell by receptor-mediated
endocytosis.
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• In the course of this process the
protein capsid and any lipoprotein
envelopes may be removed.
• Once it has entered the host cell the
viral nucleic acid is able to use the
host’s cellular machinery to synthesise
the nucleic acids and proteins required
to replicate a number of new viruses
(Fig. 10.44).
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• A great deal of information is
available concerning the details of
the mechanism of virus replication
but this text will only outline the
main points. For greater detail the
reader is referred to specialist
texts on virology.
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14.2. Classification
• RNA-viruses can be broadly classified into
two general types, namely: RNA-viruses
and RNA-retroviruses.
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• Figure 10.44 A schematic representation
of the replication ofprof.RNA-viruses
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RNA-viruses
• RNA-virus replication usually
occurs entirely in the cytoplasm.
The viral mRNA either forms
part of the RNA carried by the
virion or is synthesised by an
enzyme already present in the
virion.
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• This viral mRNA is used to
produce the necessary viral
proteins by translation using
the host cell’s ribosomes and
enzyme systems.
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• Some of the viral proteins are
enzymes that are used to catalyse
the reproduction of more viral
mRNA. The new viral RNA and viral
proteins are assembled into a
number of new virions that are
ultimately released from the host
cell by either lysis or budding.
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Retroviruses
• Retroviruses synthesise viral DNA
using their viral RNA as a template.
• This process is catalysed by enzyme
systems known as reverse
transcriptases that form part of the
virion. The viral DNA is incorporated
into the host genome to form a socalled provirus.
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• Transcription of the provirus
produces new ‘genomic’ viral RNA
and viral mRNA. The viral mRNA is
used to produce viral proteins,
which together with the ‘genomic’
viral RNA are assembled into new
virions.
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• These virions are released by
budding , which in many cases
does not kill the host cell.
Retroviruses are responsible
for some forms of cancer and
AIDS
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DNA-viruses
• Most DNA-viruses enter the host cell’s
nucleus where formation of viral mRNA
by transcription from the viral DNA is
brought about by the host cell’s
polymerases. This viral mRNA is used to
produce viral proteins by translation
using the host cell’s ribosomes and
enzyme systems.
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• Some of these proteins will be enzymes
that can catalyse the synthesis of more
viral DNA.
• This DNA and the viral proteins
synthesised in the host cell are
assembled into a number of new virions
that are ultimately released from the
host by either cell lysis or budding
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14.3. Viral diseases
• Viral infection of host cells is a common
occurrence. Most of the time this
infection does not result in illness as
the body’s immune system can usually
deal with such viral invasion.
• When illness occurs it is often short
lived and leads to long-term immunity.
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• However, a number of viral infections
can lead to serious medical conditions (.
Some viruses like HIV, the aetiological
agent of AIDS, are able to remain
dormant in the host for a number of
years before becoming active, whilst
others such as herpes zoster (shingles)
can give rise to recurrent bouts of the
illness. Both chemotherapy and
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preventative
• Both chemotherapy and preventative
vaccination are used to treat patients.
The latter is the main clinical approach
since it has been difficult to design
drugs that only target the virus.
However, a number of antiviral drugs
have been developed and are in clinical
use.
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AIDS
• AIDS is a disease that progressively
destroys the human immune system. It
is caused by the human
immunodeficiency virus (HIV), which is a
retrovirus. This virus enters and
destroys human T4 lymphocyte cells.
These cells are a vital part of the
human immune system.
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• Their destruction reduces the
body’s resistance to other
infectious diseases, such as
pneumonia, and some rare forms of
cancer.
•.
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• The entry of the virus into the body
usually causes an initial period of acute
ill health with the patient suffering
from headaches, fevers and rashes,
amongst other symptoms.
• This is followed by a period of relatively
good healthy where the virus replicates
in the lymph nodes.
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• This relatively healthy period normally
lasts a number of years before fullblown
• AIDS appears. Full-blown AIDS is
characterised by a wide variety of
diseases such as bacterial infections,
neurological diseases and cancers.
Treatment is more effective when a
mixture of antiviral agents is used
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14.4. Antiviral drugs
• It has been found that viruses utilise a
number of virus-specific enzymes during
replication.
• These enzymes and the processes they
control are significantly different from
those of the host cell to make them a
useful target for medicinal chemists.
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• Consequently, antiviral drugs
normally act by inhibiting viral
nucleic acid synthesis, inhibiting
attachment to and penetration of
the host cell or inhibiting viral
protein synthesis.
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Nucleic acid synthesis inhibitors
• Nucleic acid synthesis inhibitors usually
act by inhibiting the polymerases or
reverse transcriptases required for
nucleic acid chain formation. However,
because they are usually analogues of
the purine and pyrimidine bases found in
the viral nucleic acids, they are often
incorporated into the growing nucleic
acid chain.
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• In this case their general mode of
action frequently involves conversion to
the corresponding 50-triphosphate by
the host cell’s cellular kinases. This
conversion may also involve specific viral
enzymes in the initial
monophosphorylation step.
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• These triphosphate drug derivatives are
incorporated into the nucleic acid chain
where they terminate its formation.
Termination occurs because the drug
residues do not have the 30-hydroxy
group necessary for the phosphate
ester formation required for further
growth of the nucleic acid chain. This
effectively inhibits the polymerases and
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ranscriptases thatprof.catalyse
the growth
• This effectively inhibits the
polymerases and ranscriptases that
catalyse the growth of the nucleic
acid (Fig. 10.45).
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Aciclovir
• Aciclovir was the first effective
antiviral drug. It is effective against a
number of herpes viruses, notably
simplex, varicella-zoster (shingles),
varicella (chickenpox) and Epstein–Barr
virus (glandular fever). It may be
administered orally and by intravenous
injection as well as topically. Orally
administered doses have a low
bioavailability.
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• The action of aciclovir is more effective
in virus-infected host cells because the
viral thymidine kinase is a more
efficient catalyst for the
monophosphorylation of aciclovir than
the thymidine kinases of the host cell.
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• This leads to an increase in the
concentration of the aciclovir
triphosphate, which has 100-fold
greater affinity for viral DNA
polymerase than human DNA
polymerase.
• As a result, it preferentially
competitively inhibits viral DNA
polymerase and so prevents the virus
from replicating.
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• However, resistance has been reported
due to changes in the viral mRNA
responsible for the production of the
viral thymidine kinase. Aciclovir also
acts by terminating chain formation.
The aciclovir–DNA complex formed by
the drug also irreversibly inhibits DNA
polymerase.
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Vidarabine
• Vidarabine is active against herpes
simplex and herpes varicella-zoster.
• However, the drug does give rise to
nausea, vomiting, tremors, dizziness and
seizures. In addition it has been
reported to be mutagenic, teratogenic
and carcinogenic in animal studies.
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• Vidarabine is administered by
intravenous infusion and topical
application. It has a half-life of about
one hour, the drug being rapidly
deaminated to arabinofuranosyl
hypoxanthine (ara-HX) by adenosine
deaminase.
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• This enzyme is found in the serum and red
blood cells. Ara-HX, which also exhibits a
weak antiviral action, has a half-life of about
3.5 hours.
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Zidovudine (AZT)
• Zidovudine was originally synthesised in
1964 as an analogue of thymine by J.
Horwitz as a potential antileukaemia
drug. It was found to be unsuitable for
use in this role and for 20 years was
ignored, even though in 1974 W.
Osterag et al. reported that it was
active against Friend leukaemia virus, a
retrovirus.
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• However, the identification in 1983
of the retrovirus HIVas the source
of AIDS resulted in the virologist
M. St Clair setting up a screening
programme for drugs that could
attack HIV
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• Fourteen compounds were selected and
screened against Friend leukaemia virus
and a second retrovirus called Harvey
sarcoma virus. This screen led to the
discovery of zidovudine (AZT), which
was rapidly developed into clinical use on
selected patients in 1986.
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• AZT is converted by the action of
cellular thymidine kinase to the 50triphosphate. This inhibits the
enzyme reverse transcriptase in
the retrovirus, which effectively
prevents it from forming the viral
DNA necessary for viral replication.
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• The incorporation of AZT into the
nucleic acid chain also results in chain
termination because the presence of
the 30-azide group prevents the
reaction of the chain with the 50triphosphate of the next nucleotide
waiting to join the chain (Fig. 10.45).
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• AZT is also active against
mammalian DNA polymerase and
although its affinity for this
enzyme is about 100-fold less this
action is thought to be the cause of
some of its unwanted side effects.
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• Zidovudine is active against the
retroviruses (see section 10.14.2) that
cause AIDS (HIV virus) and certain
types of leukaemia.
• It also inhibits cellular a-DNA
polymerase but only at concentrations in
excess of 100-fold greater than those
needed to treat the viral infection.
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• The drug may be administered orally or
by intravenous infusion. The
bioavailability from oral administration
is good, the drug being distributed into
most body fluids and tissues.
• However, when used to treat AIDS it
has given rise to gastrointestinal
disorders, skin rashes, insomnia,
anaemia, fever, headaches, depression
and other unwanted effects.
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Resistance
• Resistance increases with time.
This is known to be due to the virus
developing mutations’ which result
in changes in the amino acid
sequences in the reverse
transcriptase.
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Didanosine
• Didanosine is used to treat some AZT-
resistant strains of HIV. It is also used
in combination with AZT to treat HIV.
Didanosine is administered orally in
dosage forms that contain antacid
buffers to prevent conversion by the
stomach acids to hypoxanthine
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• However, in spite of the use of buffers
the bioavailability from oral
administration is low.
• The drug can cause nausea, abdominal
pain and peripheral neuropathy, amongst
other symptoms. Drug resistance occurs
after prolonged use.
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• Didanosine is converted by viral and
cellular kinases to the monophosphate
and then to the triphosphate. In this
form it inhibits reverse transcriptase
and in addition its incorporation into the
DNA chain terminates the chain
because the drug has no 30-hydroxy
group (Fig. 10.45).
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Host cell penetration inhibitors
• The principal drugs that act in this
manner are amantadine and rimantadine
(Fig. 10.46).
• Both amantadine and rimantadine are
also used to treat Parkinson’s disease.
However, their mode of action in this
disease is different from their action
as antiviral agents.
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Amantadine hydrochloride
• Amantadine hydrochloride is
effective against influenza A virus
but is not effective against the
influenza B virus. When used as a
prophylactic, it is believed to give
up to 80 per cent protection
against influenza A virus infections
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• The drug acts by blocking an ion
channel in the virus membrane
formed by the viral proteinM2. This
is believed to inhibit the
disassembly of the core of the
virion and its penetration of the
host (see section 10.14.1).
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• Amantadine hydrochloride has a good
bioavailability on oral administration,
being readily absorbed and distributed
to most body fluids and tissues.
• Its elimination time is 12–18 hours.
However, its use can result in
depression, dizziness, insomnia and
gastrointestinal disturbances, amongst
other unwanted side effects.
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Rimantadine hydrochloride
• Rimantadine hydrochloride is an
analogue of amantadine
hydrochloride. It is more effective
against influenza A virus than
amantadine. Its mode of action is
probably similar to that of
amantadine.
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• The drug is readily absorbed when
administered orally but undergoes
extensive first-pass metabolism.
However, in spite of this, its
elimination half-life is double that
of amantadine. Furthermore, CNS
side effects are significantly
reduced.
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Inhibitors of viral protein
synthesis
• The principal compounds that act as
inhibitors of protein synthesis are the
interferons.
• These compounds are members of a
naturally occurring family of
glycoprotein hormones (RMM 20 000–
160 000), which are produced by nearly
all types of eukaryotic cell.
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• Three general classes of interferons
are known to occur naturally in
mammals, namely: the α-interferons
produced by leucocytes, β-interferons
produced by fibroblasts and γinterferons produced by T lymphocytes.
At least twenty α-, two β- and two γinterferons have been identified
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• Interferons form part of the human
immune system. It is believed that the
presence of virions, bacteria and other
antigens in the body switches on the
mRNA that controls the production and
release of interferon. This release
stimulates other cells to produce and
• release more interferon.
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• Interferons are thought to act by
initiating the production in the cell
of proteins that protect the cells
from viral attack. The main action
of these proteins takes the form of
inhibiting the synthesis of viral
mRNA and viral protein synthesis.
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• a- Interferons also enhance the
activity of killer T cells
associated with the immune
system. (see section 14.5.5).
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• The main action of these proteins
takes the form of inhibiting the
synthesis of viral mRNA and viral
protein synthesis.
• α- Interferons also enhance the
activity of killer T cells associated
with the immune system.
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• A number of a-interferons have
been manufactured and proven to
be reasonably effective against a
number of viruses and cancers.
• Interferons are usually given by
intravenous, intramuscular or
subcutaneous injection.
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• However, their administration can cause
adverse effects, such as headaches,
fevers and bone marrow depression,
that are dose related.
• The formation and release of interferon
by viral and other pathological
stimulation has resulted in a search for
chemical inducers of endogenous
interferon.
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• Administration of a wide range of
compounds has resulted in the
induction of interferon production.
However, no clinically useful
compounds have been found for
humans’ although tilorone is
effective in inducing interferon in
mice.
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10.15. Recombinant DNA (Genetic
Engineering)
• The body requires a constant supply of
certain peptides and proteins if it is to
remain health and function normally.
Many of these peptides and proteins are
only produced in a small quantities. They
will be produced only if the correct
genes are present in the cell.
Consequently, if a gene is missing or
defective an essential protein will
• not be produced, which can lead to a
diseased state.
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• Consequently, if a gene is
missing or defective an
essential protein will not be
produced, which can lead to a
diseased state.
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• For example, cystic fibrosis is
caused by a defective gene. This
faulty gene produces a defective
membrane protein, cystic fibrosis
transmembrane regulator (CFTR),
which will not allow the free
passage of chloride ions through
the membrane
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• The passage of chloride ions through a
normal membrane into the lungs is
usually accompanied by a flow of water
molecules in the same direction.
• In membranes that contain CFTR the
transport of water through the
membrane into the lungs is reduced.
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• This viscous mucus clogs the lungs
and makes breathing difficult, a
classic symptom of cystic fibrosis.
It also provides a breeding ground
for bacteria that cause pneumonia
and other illnesses.
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• Several thousand hereditary diseases
found in humans are known to be caused
by faulty genes. Recombinant DNA
(rDNA) technology (genetic engineering)
offers a new way of combating these
hereditary diseases by either replacing
the faulty genes or producing the
missing peptides and proteins so that
they can be given as a medicine (see
section 10.15.2).
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• The first step in any use of
recombinant DNA technology is to
isolate or copy the required gene.
There are three sources of the
genes required for cloning. The two
most important are genomic and
copy or complementary DNA
(cDNA) libraries.
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• In the first case the library
consists of DNA fragments
obtained from a cell’s genome,
whilst in the second case the
library consists of DNA fragments
synthesised by using the mRNA for
the protein of interest.
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• The third is by the automated
synthesis of DNA, which is only
feasible if the required base
sequence is known. This may be
deduced from the amino acid
sequence of the required protein if
it is known.
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• Once the gene has been obtained it
is inserted into a carrier (vector)
that can enter a host cell and be
replicated, propagated and
transcripted into mRNA by the
cellular biochemistry of that cell.
This process is often referred to
as gene cloning.
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• The mRNA produced by the cloned DNA
is used by the cell ribosomes to produce
the protein encoded by the cloned DNA.
In theory, gene cloning makes it possible
to produce any protein provided that it
is possible to obtain a copy of the
corresponding gene. Products produced
using recombinant DNA usually have
recombinant, r or rDNA in their names.
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15.1. Gene cloning
• Bacteria are frequently used as
host cells for gene cloning. This is
because they normally use the same
genetic code as humans to make
peptides and proteins. However, in
bacteria the mechanism for peptide
and protein formation is somewhat
different.
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• It is not restricted to the
chromosomes but can also occur in
extranuclear particles called
plasmids. Plasmids are large circular
supercoiled DNA molecules whose
structure contains at least one gene
and a start site for replication.
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• However, the number of genes
found in a plasmid is fairly limited,
although bacteria will contain a
number of identical copies of the
same plasmid.
• It is possible to isolate the
plasmids of bacterial cells.
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• The isolated DNA molecules can
bebroken open by cleaving the
phosphate bonds between specific
pairs of bases by the action
enzymes known as restriction
enzymes or endonucleases
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• Each of these enzymes, of which
over 500 are known, will only cleave
the bonds between specific
nucleosides. For example, EcoR I
cleaves the phosphate link between
guanosine and adenosine whilst Xho
I cuts the chain between cytidine
and thymine nucleosides.
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• Cutting the strand can result in
either blunt ends, where the
endonuclease cuts across both
chains of the DNA at the same
points, or cohesive ends (sticky
ends), where the cut is staggered
from one chain to the other (Fig.
10.47).
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• The new non-cyclic structure of the
plasmid is known as linearised DNA
in order to distinguish it from the
new insert or foreign DNA.
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• This foreign DNA must contain the
required gene, a second gene system
that confers resistance to a specific
antibiotic and any other necessary
information. It should be remembered
that a eukaryotic gene is made up of
exons separated by introns, which are
sequences that have no apparent use.
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• Figure 10.47 (a) Blunt and (b) cohesive
cuts with compatible adhesive cuts
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• Mixing the foreign DNA and the
linearised DNA in a suitable medium
results in the formation of extended
plasmid loops when their ends come into
contact (Fig. 10.48). This contact is
converted into a permanent bond by the
catalytic action of an enzyme called
DNA ligase.
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• Figure
10.48. A
representa
tion of the
main steps
in the
insertion
of a gene
into a
plasmid
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• This contact is converted into a
permanent bond by the catalytic
action of an enzyme called DNA
ligase. When the chains are
cohesive the exposed single chains
of new DNA must contain a
complementary base sequence to
the exposed ends of the linearised
DNA.
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• The hydrogen bonding between these
complementary base pairs tends to bind
the chains together prior to the action
of the DNA ligase, hence the name
‘‘sticky ends’’. The new DNA of the
modified plasmid is known as
recombinant DNA (rDNA).
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• However, the random nature of the
techniques used to form the modified
plasmids means that some of the
linearised DNA reforms the plasmid
without incorporating the foreign DNA,
that is, a mixture of both types of
plasmid is formed.
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• The modified plasmids are
separated from the unmodified
plasmids when they are reinserted
into a bacterial cell.
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• The new plasmids are reinserted into
the bacteria by a process known as
transformation.
• Bacteria are mixed with the new
plasmids in a medium containing calcium
chloride. This medium makes the
bacterial membrane permeable to the
plasmid.
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• However, not all bacteria will take
up the modified plasmids. Such
bacteria can easily be destroyed by
specific antibiotic action since they
do not contain plasmids with the
appropriate protecting gene.
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• This makes isolation of the bacteria
with the modified plasmids
relatively simple.
• These modified bacteria are
allowed to replicate and, in doing so,
produce many copies of the
modified plasmid.
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• Under favourable conditions one
modified bacterial cell can produce
over 200 copies of the new plasmid.
The gene in these modified
plasmids will use the bacteria’s
internal machinery to automatically
produce the appropriate peptide or
protein.
prof. aza
• Since many bacteria replicate at a
very rapid rate this technique
offers a relatively quick way of
producing large quantities of
essential naturally occurring
compounds that cannot be produced
by other means.
prof. aza
• Plasmids are not the only vectors
that can be used to transport DNA
into a bacterial host cell.
• Foreign DNA can also be inserted
into bacteriophages and cosmids by
similar techniques.
prof. aza
• Bacteriophages (phage) are viruses that
specifically infect bacteria whilst a
cosmid is a hybrid between a phage and
a plasmid that has been especially
synthesised for use in gene cloning.
Plasmids can be used to insert
fragments containing up to 10
kilobasepairs (kbp), phages up to 20 kbp
and cosmids 50 or more kbp.
prof. aza
• It is not always necessary to use a
vector to place the recombinant
DNA in a cell. If the cell is large
enough, the recombinant DNA may
be placed in the cell by using a
micropipette whose overall tip
diameter is less than 1 mm.
prof. aza
• Only a small amount of the
recombinant DNA inserted in this
fashion is taken up by the cell’s
chromosomes. However, this small
fraction will increase to a
significant level as the cell
replicates (Fig. 10.48).
prof. aza
• Host cells for all methods of cloning are
usually either bacterial or mammalian in
origin. For example, bacterial cells often
used are E. coli and eukaryotic yeast
while mammalian cell lines include
Chinese hamster ovary (CHO), baby
hamster kidney (BHK) and African green
monkey kidney (VERO).
prof. aza
• In all cases small-scale cultures of
the host cell plus vector are grown
to find the culture containing the
host with the required gene that
gives the best yield of the desired
protein.
prof. aza
• Once this culture has been
determined the process is scaled up
via a suitable pilot plant to
production level (see section 16.6).
The mammalian cell line cultures
normally give poorer yields of the
desired protein.
prof. aza
15.2.2 Manufacture of Pharmaceuticals
• The body produces peptides and
proteins, often in extremely small
quantities, which are essential for its
well being. The absence of the
necessary’ genes means that the body
does not produce these essential
compounds, resulting in a deficiency
disease that is usually’ fatal.
• Treatment by supplying the patient with
sufficient amounts of the missing
compounds is normally successful.
prof. aza
• However, extraction from other
natural sources is usually’ difficult
and yields are often low. For
example, it takes half a million
sheep brains to produce 5mg of
somatostatin a growth hormone
that inhibits secretion of the
pituitary growth hormone.
prof. aza
• Furthermore, unless the source of
the required product is donated
blood there is a limit to the number
of cadavers available for the
extraction of compounds suitable
for use in humans.
prof. aza
• Moreover, there is also the danger that
compounds obtained from human
sources may be contaminated by’ viruses
such as HIV, hepatitis, Creutzfeld–
Jakob disease (mad cow disease) and
others that are difficult to detect.
Animal sources have been used but only
a few human protein deficiency
disorders can be treated with animal
proteins.
prof. aza
• Gene cloning is used to obtain human
recombinant proteins. However, some
proteins will also need post—
translational modification such as
glycosylation and/or the modification of
amino acid sequences. These
modifications may require forming
different section, of the peptide chain
in the culture medium and chemically’
combining these sections in vitro.
prof. aza
• The genes required for these processes
are synthesised using the required peptide
as a blueprint. For example, human
recombinant insuline may he produced in
this manner (Figure 10.12). The genes for
the A and B chains of insulin were
synthesised separately.
prof. aza
• They were cloned separately, using
suitable plasmids. into two different
bacterial strains. One of these strains
is used to produce the A chain whilst
the others is used to produce the B
strain. The chains are isolated and
attached to each other by in vitro
disulphide bond formation.
prof. aza
• This last step is inefficient and
human recombinant insulin is now
made by forming recombinant
proinsulin by gene cloning. The
proinsulin is converted to
recombinant insulin by proteolytic
cleavage
prof. aza
Figure 10.42. An outline of the synthesis of
recombinant human insulin.
prof. aza